This is a revision application of 5R33CA099139-04 Parallel Peptide Tandem Mass Spectrometry to the program NOT-OD-09-058 NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The original R33 award was devised around a method to increase protein identifications by fragmenting all peptides present in a wide (e.g. 400-1400) m/z range. The idea being that this shotgun CID (or multiplexed collision induced dissociation is more efficient than standard shotgun proteomic methods that use dad-dependent ion selection during HPLC introduction of the sample to a tandem mass spectrometer. While this approach worked, the efficiency was offset by the degeneracy in many of the identifications. This led us to modify our basic data acquisition approach. Instead of looking at all peptides in a wide m/z range, we carried out an experiment to look at all peptides in each m/z "channel". Obviously, this requires many more injections, but as described below the approach worked remarkably well to increase the number of confident protein identifications, protein sequence coverage and dynamic range all of which was achieved without prior sample fractionation. The downside is the time it takes to acquire data set, typically five days of continuous automated LC-MS/MS time. Here we request a one year extension of our R33 to optimize a method we refer to as Peptide Acquisition Independent From Ion Count (PAcIFIC) by: Aim 1) Optimize Peptide Acquisition Independent From Ion Count (PAcIFIC) making it quantitative and multiplexed as well as reducing acquisition time to two days, Aim 2) Use Precursor Ion Independent Top- down Algorithm (PIITA) to identify "small" proteins by modification of wet and dry lab methods as well as to incorporate PAcIFIC and Aim 3) Identify and eliminate sources of chemical noise, PAcIFIC data sets being systematically acquired on each and every m/z channel allows a thorough understanding of all peptides/proteins detectable (from low and high signal/noise) and how sources of non-peptide "noise" may be diminished to aid detection of low abundance proteins. PUBLIC HEALTH RELEVANCE: A unifying goal of many NIH programs is to drastically reduce deaths from cancer. The current thinking is this may be achieved through early detection of markers of disease;e.g. proteins and metabolites. Preliminary data supporting our proposal shows that new proteins may be identified by simple modifications to the way proteomic data is acquired and analyzed. Our proposed plan will lead to more facile methods for protein discovery that may be immediately adopted by laboratories searching for protein markers of disease.